Journal: Cells

Article Title: ELISPOTs Produced by CD8 and CD4 Cells Follow Log Normal Size Distribution Permitting Objective Counting

doi: 10.3390/cells4010056

Figure Lengend Snippet: Log Normal size distributions for cytokine ELISPOTs produced by CD8 and CD4 cells. ( A ) For CD8 cells, IFN-γ ELISPOT size distributions were studied for a total of 334 positive recall responses induced by the 32 individual CEF peptides. Each peptide is represented by an individual symbol type. ( B – F ) For CD4 cells, 80 individual IFN-γ, IL-2, IL- 4, IL-5, and IL-17 spot size distributions elicited by inactivated CMV and EBV were studied; each one specified by a different symbol. The Kolmogorov–Smirnov goodness of fit test was used to determine the normality of spot size distribution for each individual positive recall response. Experimental IDs of the positive donors are shown on the x-axis, and the y-axis represents the p-values for the donor/peptide combinations. The red line indicates the cut-off significance level of 5%.

Article Snippet: Human Interferon-γ ImmunoSpot® kits (CTL-HIFNG-1/5M), human IL-2- (CTL-HIL21M/5), IL-4- (CTL-HIL4-1M/5), IL-5- (CTL-HIL5-1M/5), and IL-17 kits (CTL-HIL17-1M/5) were obtained from CTL.

Techniques: Produced, Enzyme-linked Immunospot

Journal: Cells

Article Title: ELISPOTs Produced by CD8 and CD4 Cells Follow Log Normal Size Distribution Permitting Objective Counting

doi: 10.3390/cells4010056

Figure Lengend Snippet: ( A ) IFN-γ, ( B ) IL-2, ( C ) IL-4, ( D ) IL-5, and ( E ) IL-17 ELISPOTs produced by CD4 cells show a maximal three-fold size variation between donors and antigens. Inactivated EBV or CMV virions (indicated by the different symbols) were used to induce CD4 cells of different donors to produce the cytokines specified in the panels. The mean spot size (mm 2 ) and SD for each positive response (y-axis) are plotted in the logarithmic scale vs. the individual donors’ ID specified on the x-axis. For each data point 1000 spots were analyzed.

Article Snippet: Human Interferon-γ ImmunoSpot® kits (CTL-HIFNG-1/5M), human IL-2- (CTL-HIL21M/5), IL-4- (CTL-HIL4-1M/5), IL-5- (CTL-HIL5-1M/5), and IL-17 kits (CTL-HIL17-1M/5) were obtained from CTL.

Techniques: Produced

Journal: Frontiers in Immunology

Article Title: Selective Engagement of FcγRIV by a M2e-Specific Single Domain Antibody Construct Protects Against Influenza A Virus Infection

doi: 10.3389/fimmu.2019.02920

Figure Lengend Snippet: Bispecific fusion construct of anti-mouse FcγRIV VHH with M2e-VHH-23m selectively activates FcγRIV in vitro . (A) Schematic representation of the bispecific VHHs and ELISA on human H3N2 M2e peptide and recombinant FcγRIV protein is shown on the right. Wells of microtiter plates were coated with 100 ng peptide or protein. Dilution series of the bispecific VHH fusion constructs were added to the coated plates. Binding was detected with a mouse anti-His tag MAb, followed by a secondary sheep anti-mouse IgG Ab conjugated to HRP for the peptide ELISA. In the ELISA with coated recombinant FcγRIV protein, binding was detected with a HRP-conjugated rabbit anti-camelid VHH antibody. (B) Serial dilutions of the bispecific VHH fusion construct or monoclonal antibodies were added to HEK293T cells stably transfected with an influenza M2 expression plasmid. Thirty minutes later, FcγR-ζ BW5147 reporter cells were added to the HEK293T cells. After overnight incubation produced mIL-2 was measured in a sandwich-ELISA, which served as an indicator for the magnitude of FcγR activation. (C) MDCK cells were infected with A/Puerto Rico/8/1934 (H1N1) virus for 1 h. Unbound virus particles were washed away and serial dilutions of the bispecific VHHs or monoclonal antibodies were added and incubated for 30 min, followed by the addition of the FcγRIV-ζ BW5147 reporter cells (C) or human FcγRIIIa-ζ BW5147 reporter cells (D) . After overnight incubation supernatants were analyzed by an anti mIL-2 sandwich ELISA. Data points represent averages of triplicates and error bars represent standard deviations. The graphs are a representative of one out of three repeat experiments.

Article Snippet: Supernatants were analyzed by an anti-mIL-2 sandwich ELISA as described using the capture MAb JES6-1A12 and the biotinylated detection MAb JES6-5H4 (BD Pharmingen™, Belgium) ( , ).

Techniques: Construct, In Vitro, Enzyme-linked Immunosorbent Assay, Recombinant, Binding Assay, Peptide ELISA, Protein Binding, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Incubation, Produced, Sandwich ELISA, Activation Assay, Infection